9405733 Kustu The NIFA protein (nifA gene product) is required to activate transcription of the nitrogen fixation (nif) operons in a wide variety of free-living and symbiotic bacteria belonging to the large gram-negative division proteobacteria. To activate, NIFA binds to enhancer-like sites approximately 100 bp upstream of nif promoters and allows RNA polymerase (alpha54-holoenzyme form) to denature the DNA strands around a transcriptional startsite -- that is, to isomerize from closed to open complexes. We succeeded in purifying the insoluble NIFA protein from Klebsiella pneumoniae as a fusion to the soluble maltose-binding protein (MBP) and in demonstrating its binding to enhancer-like sites in vitro for the first time. MBP-NIFA activated transcription by alpha54- holoenzyme in a purified system and could be shown to catalyze the isomerization of closed complexes between this polymerase and the nifH promoter to open complexes. Activation and open complex formation required a nucleoside triphosphate with a hydrolyzable, beta-gamma bond but we were unable to demonstrate hydrolysis directly because we could not remove contaminating hydrolytic activities from the preparation. We also purified an MBP fusion to just the central catalytic domain of NIFA, in the absence of its DNA-binding domain, and released the central domain in a soluble, active form by proteolytic cleavage of the fusion protein . The released central domain could activate transcription from solution, a property so far unique among enhancer-binding proteins, and had the expected ability to hydrolyze nucleotides. A purified renatured form of the NIFL protein, which is known to inhibit NIFA activity in vivo in response to the presence of molecular oxygen or combined nitrogen, inhibited transcriptional activation by both MBP-NIFA and the central domain of NIFA. Since NIFL did not inhibit nucleotide hydrolysis by the central domain, we postulate that it interferes with protein-protein contact between this domain of NIFA and alpha54-holoenzyme. Our major goals for the next grant period are: 1) to obtain NIFL preparations with better activity; 2) to determine whether NIFL inhibits NIFA activity by interacting with NIFA stoichiometrically or covalently modifying it; 3) to study protein-protein interactions between NIFL and NIFA and between NIFA and polymerase; and 4) to determine the mechanism by which NIFL senses molecular oxygen. The studies are of interest with respect to understanding the interaction of enhancerbinding proteins with the* target RNA polymerases and with regard to understanding the various forms of regulation that occur in response to molecular oxygen. We hope that they will eventually be of use to others in increasing the efficiency of biological nitrogen fixation. %%% Crop productivity is often limited by the availability of nitrogen in a fornm suitable for synthesis of proteins and other large molecules characteristie of living organisms. (certain bacteria, eithel alone or in partnership with plants, have the ability lo convert nitrogen gas from the atmosphere to ammonia, a nitrogen fertilizer, in a process called biological nitrogen fixation. We are studying the NIFA protein, a major regulator of biological nitrogen fixation. Characterizing NlFA will contnbute to understanding, how organisms decode palticu ar portions of their DNA under appropriate conditions and may help to improve the biological productivn of nitrogen felti1izer. A major goal for the next grant period is to understand how the function of the NIFA protein is poisoned by oxygen gas in the air. ***
